Polymerase chain reaction (PCR) is a method whereby virtually any DNA sequence can be selectively amplified. The method involves using paired sets of oligonucleotides of predetermined sequence that hybridize to opposite strands of DNA and define the limits of the sequence to be amplified. The oligonucleotides prime multiple sequential rounds of DNA synthesis catalyzed by a thermostable DNA polymerase. Each round of synthesis is typically separated by a melting and re-annealing step, allowing a given DNA sequence to be amplified several hundred-fold in less than an hour (Saiki et al., Science 239:487, 1988).
With the rapid advances in mammalian molecular genetics, an ever increasing number of disease genes have been identified. Accordingly, PCR has gained widespread use for the diagnosis of inherited disorders and the susceptibility to disease. Typically, the region of interest is amplified from either genomic DNA or from a source of specific cDNA encoding the cognate gene product. Mutations or polymorphisms are then identified by subjecting the amplified DNA to analytical techniques such as DNA sequencing, hybridization with allele-specific oligonucleotides (ASOs), oligonucleotide ligation, restriction endonuclease cleavage or single-strand conformational polymorphism (SSCP) analysis.
For the analysis of small genes and transcripts, or genes where the mutant allele or polymorphism is well characterized, amplification of a single defined region of DNA is sometimes sufficient. When analyzing large genes and transcripts or undefined genes, however, multiple individual PCR reactions are often required to identify critical base changes or deletions. Thus, to streamline the analysis of large complex genes, multiplex PCR (i.e., the simultaneous amplification of different target DNA sequences in a single PCR reaction) has been utilized.
The results obtained with multiplex PCR are, however, frequently complicated by artifacts of the amplification procedure. These include "false-negative" results due to reaction failure and "false-positive" results such as the amplification of spurious products, which may be caused by annealing of the primers to sequences which are related to, but distinct from, the true recognition sequences.
For use in multiplex PCR, a primer should be designed so that its predicted hybridization kinetics are similar to those of the other primers used in the same multiplex reaction. While the annealing temperatures and primer concentrations may be calculated to some degree, conditions generally have to be empirically determined for each multiplex reaction. Since the possibility of non-specific priming increases with each additional primer pair, conditions must be modified as necessary as individual primer sets are added. Moreover, artifacts that result from competition for resources (e.g., depletion of primers) are augmented in multiplex PCR, since differences in the yields of unequally amplified fragments are enhanced with each cycle.
Weighardt et al. (PCR Meth.App. 3:77, 1993) describe the use of 5'-tailed oligonucleotides for PCR. However, a key feature of this amplification method involves separate annealing and primer extension reactions for each primer, which is not practical in a multiplex context. Therefore, complete optimization of the reaction conditions for multiplex PCR can become labor intensive and time consuming. Since different multiplex PCRs may each have unique reaction conditions, development of new diagnostic tests can become very costly.
Thus, there is a need in the art for primers that allow multiplex PCR reactions to be designed and carried out without elaborate optimization steps, irrespective of the potentially divergent properties of the different primers used. Furthermore, there is a need in the art for primers that allow multiplex PCR reactions that, under the same reaction conditions, simultaneously produce equivalent amounts of each of many amplification products.